Whisker-mediated transformation of cotton embryogenic callus tissues and regeneration of plants thereof

ABSTRACT

Cotton embryogenic callus tissues can be transformed by elongated, needle-like structures called “whiskers”. The process comprises the agitation of plant cell aggregates and plant tissues of the plant to be transformed in the presence of DNA and whiskers, whereby DNA uptake and integration thereof is facilitated.

RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 09/239,706, filed Jan.28, 1999, now U.S. Pat. No. 6,350,611, which was entitled to priorityfrom U.S. Ser. No. 60/072,944, filed Jan. 29, 1998.

FIELD OF INVENTION

This invention relates to a method of using elongated, needle-likemicrofibers or “whiskers” to transform plant cell aggregates andselected plant tissues.

BACKGROUND OF THE INVENTION

Until recently, genetically manipulated plants were limited almostexclusively to those events created by application of classical breedingmethods. Creation of new plant varieties by breeding was reservedprimarily for the most agronomically important crops, such as corn, dueto the cost and time needed to identify, cross, and stably fix a gene inthe genome, thus creating the desired trait. In comparison, the adventof genetic engineering has resulted in the introduction of manydifferent heterologous genes and subsequent traits into diverse cropsincluding corn, cotton, soybeans, wheat, rice, sunflowers and canola ina more rapid manner. However, the intergression of a new transgene intoelite germplasm is still quite a laborious task due to the tissueculturing and back-crossing needed to produce a commercially viable,elite, line.

Several techniques exist which allow for the introduction, plantregeneration, stable integration, and expression of foreign recombinantvectors containing heterologous genes of interest in plant cells. Onesuch technique involves acceleration of microparticles coated withgenetic material directly into plant cells (U.S. Pat. Nos. 4,945,050 toCornell; 5,141,131 to DowElanco; and 5,538,877 and 5,538,880, both toDekalb). This technique is commonly referred to as “microparticlebombardment” or “biolistics”. Plants may also be transformed usingAgrobacterium technology (U.S. Pat. No. 5,177,010 to University ofToledo, 5,104,310 to Texas A&M, European Patent Application 0131624B1,European Patent Applications 120516, 159418B1 and 176,112 toSchilperoot, U.S. Pat. Nos. 5,149,645, 5,469,976, 5,464,763 and4,940,838 and 4,693,976 to Schilperoot, European Patent Applications116718, 290799, 320500 all to Max Planck, European Patent Applications604662,627752 and U.S. Pat. No. 5,591,616 to Japan Tobacco, EuropeanPatent Applications 0267159, and 0292435 and U.S. Pat. No. 5,231,019 allto Ciba-Geigy, U.S. Pat. Nos. 5,463,174 and 4,762,785 both to Calgene,and U.S. Pat. Nos. 5,004,863 and 5,159,135 both to Agracetus). Anothertransformation method involves the use of elongated needle-likemicrofibers or “whiskers” to transform cell suspension cultures (U.S.Pat. Nos. 5,302,523 and 5,464,765 both to Zeneca). In addition,electroporation technology has been used to transform plant cells fromwhich fertile plants have been obtained (WO 87/06614 to Boyce ThompsonInstitute; U.S. Pat. Nos. 5,472,869 and 5,384,253 both to Dekalb; U.S.Pat. Nos. 5,679,558, 5,641,664, WO9209696 and WO9321335 to Plant GeneticSystems).

Despite all of the technical achievements, genetic transformation androutine production of transgenic plants in a commercially viable, elite,germplasm is still a laborious task. For example, microparticlebombardment, while capable of being used either on individual cells,cell aggregates, or plant tissues, requires preparing DNA-attached goldparticles and optimization of an expensive and not yet widely available,“gun” apparatus. Techniques involving Agrobacterium are extremelylimited because not all plant species or varieties within a givenspecies are susceptible to infection by the bacterium. Electroporationtechniques are not preferred due to the extreme difficulties and costtypically encountered in routinely making protoplast from differentplant species and tissues thereof and the concomitant low viability andlow transformation rate associated therewith.

As disclosed herein, applicants have invented a method whereby plantcell aggregates and plant tissues from non-elite and elite germplasm canbe directly and inexpensively transformed with a recombinant vectorcontaining the gene of choice using whiskers. Applicants' invention isadvantageous over currently used methods in that it is simple, quick andeasy to use. Furthermore, applicants' invention is superior to thatdescribed in the art in that it eliminates the need to establish TypeIII callus cultures or establish and maintain cell suspension cultures,and can be used with either Type I or Type II callus, thus, is lessgermplasm limited. This means that applicants' invention, as describedherein, can be used to transform elite genotypes directly thuseliminating the problems and time generally associated with geneintergression.

SUMMARY OF THE INVENTION

The present invention relates to the production of fertile, transgenic,Zea mays plants containing heterologous DNA preferably integrated intothe chromosome of said plant and heritable by the progeny thereof.

One aspect of the present invention relates to Zea mays plants, plantparts, plant cells, plant cell aggregates, and seed derived fromtransgenic plants containing said heterologous DNA.

The present invention also relates to the production of fertile,transgenic, Oryza sativa L. plants containing heterologous DNApreferably integrated into the chromosome of said plant and heritable bythe progeny thereof.

Another aspect of the present invention relates to Oryza sativa L.plants, plant parts, plant cells, plant cell aggregates, and seedderived from transgenic plants containing said heterologous DNA.

The present invention also relates to the production of fertile,transgenic, Gossypium hirsutum L. plants containing heterologous DNApreferably integrated into the chromosome of said plant and heritable bythe progeny thereof.

Another aspect of the present invention relates to Gossypium hirsutum L.plants, plant parts, plant fibers, plant cells, plant cell aggregates,and seed derived from transgenic plants containing said heterologousDNA.

The invention further relates to a process for producing fertiletransformed plants from Type I callus, Type II callus, hypocotyl-derivedcallus, or cotyledon-derived callus by whisker-mediated transformation.

The invention yet further relates to a process for producing fertiletransformed plants from meristematic tissue by whisker-mediatedtransformation.

Another aspect of the invention relates to fertile, mature maize plantsregenerated from Type I or Type II callus and transgenic seed producedtherefrom.

Another aspect of the invention relates to fertile, mature rice plantsregenerated from Type I callus and transgenic seed produced therefrom.

Yet, another aspect of the invention relates to fertile, mature cottonplants regenerated from hypocotyl-derived callus or cotyledon-derivedcallus and transgenic seed and fiber produced therefrom.

In a preferred embodiment, this invention produces the fertiletransgenic plants described herein by means of whisker-mediated cellperforation and heterologous DNA uptake, said whisker-mediated cellperforation being performed on plant cell aggregates and plant tissues,followed by a controlled regimen for selection and production oftransformed plant lines.

Other aspects, embodiments, advantages, and features of the presentinvention will become apparent from the following specification.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods for production of fertiletransgenic plants and seeds of, for example, the species Zea mays, Oryzasativa L., Gossypium hirsutum L., and Brassica napus by transformingplant cell aggregates and plant tissues of said species with the DNAconstruct of interest via whisker-mediated transformation. Aftertransformation, transgenic plants are regenerated from said transformedplant cell aggregates and plant tissues and said regenerated plantsexpress the chimeric DNA construct of interest. The transgenic plantsproduced herein by the methods described include: all species of cornincluding but not limited to field corn, popcorn, sweet corn, flintcorn, dent corn and the like; all species of cotton; and all species ofrice.

The following phrases and terms are defined below:

By “antisense” is meant an RNA transcript that comprises sequencescomplementary to a target RNA and/or mRNA or portions thereof and thatblocks the expression of a target gene by interfering with theprocessing, transport, and/or translation of its primary transcriptand/or mRNA. The complementarity may exist with any part of the targetRNA, i.e., the 5′ non-coding sequence, 3′ non-coding sequence, introns,or the coding sequence. Antisense RNA is typically a complement (mirrorimage) of the sense RNA.

By “cDNA” is meant DNA that is complementary to and derived from a mRNA.

By “chimeric DNA construction” is meant a recombinant DNA containinggenes or portions thereof from one or more species in either the senseor antisense orientation.

By “constitutive promoter” is meant promoter elements that directcontinuous gene expression in all cell types and at all times (i.e.,actin, ubiquitin, CaMV 35S, 35T, and the like).

By “cosuppression” is meant the introduction of a foreign gene havingsubstantial homology to an endogenous gene, and in a plant cell causesthe reduction in activity of the foreign gene and/or the endogenous geneproduct. Cosuppression can be sometimes achieved by introducing intosaid plant cell either the promoter sequence, the 5′ and/or 3′ ends,introns or the coding region of a gene.

By “developmental specific” promoter is meant promoter elementsresponsible for gene expression at specific plant developmental stages,such as in early or late embryogenesis and the like.

By “enhancer” is meant nucleotide sequence elements which can stimulatepromoter activity such as those from maize streak virus (MSV), alfalfamosaic virus (AMV), alcohol dehydrogenase intron 1 and the like.

By “expression” as used herein, is meant the transcription of enzymaticnucleic acid molecules, mRNA, and/or the antisense RNA inside a plantcell. Expression of genes also involves transcription of the gene andmay or may not involve translation of the mRNA into precursor or matureproteins.

By “foreign” or “heterologous gene” is meant a gene having a DNAsequence that is not normally found in the host cell, but is introducedby whisker-mediated transformation.

By “gene” is meant to include all genetic material involved in proteinexpression including chimeric DNA constructions, genes, plant genes andportions thereof.

By “genome” is meant genetic material contained in each cell of anorganism and/or virus.

By “inducible promoter” is meant promoter elements which are responsiblefor expression of genes in response to a specific signal, such as:physical stimuli (heat shock genes); light (RUBP carboxylase); hormone(Em); metabolites, stress and the like.

By “modified plant” is meant a plant wherein the mRNA levels, proteinlevels or enzyme specific activity of a particular protein have beenaltered relative to that seen in an unmodified plant. Modification canbe achieved by methods such as antisense, cosuppression, orover-expression.

By “plant cell aggregates”, “plant cell lines”, and “callus cell lines”is meant proliferating masses of tissue composed of a combination ofundifferentiated and differentiated cells undergoing de novomorphogenesis and formed by placing a piece of plant material (explant)onto a growth-supporting medium under sterile conditions. The terms“plant cell aggregates”, “plant cell lines”, and “callus cell lines” aremeant to include Type I and Type II callus cultures in monocotyledonousplants and hypocotyl- and cotyledon derived cultures in dicotyledonousplants. The terms defined herein are not intended to include eitherplant cell suspension cultures or Type III callus cultures.

By “plant tissues” is meant organized tissues including but not limitedto meristems, embryos, pollen, cotyledons, germ cells, and the like.

By “promoter regulatory element” is meant nucleotide sequence elementswithin a nucleic acid fragment or gene which controls the expression ofthat nucleic acid fragment or gene. Promoter sequences provide therecognition for RNA polymerase and other transcriptional factorsrequired for efficient transcription. Promoter regulatory elements froma variety of sources can be used efficiently in plant cells to expresssense and antisense gene constructs. Promoter regulatory elements arealso meant to include constitutive promoters, tissue-specific promoters,developmental-specific promoters, inducible promoters and the like.Promoter regulatory elements may also include certain enhancer sequenceelements that improve transcriptional or translational efficiency.

By “tissue-specific” promoter is meant promoter elements responsible forgene expression in specific cell or tissue types, such as the leaves orseeds (i.e., zein, oleosin, napin, ACP, globulin and the like).

By “transgenic” is meant to include any Type I or Type II callus,hypocotyl- or cotyledon derived callus, tissue, plant parts or plantswhich contains heterologous DNA or a chimeric gene construct that wasintroduced into said callus, tissue, plant parts or plants by whiskersand was subsequently transferred to later generations by sexual orasexual cell crosses or cell divisions.

By “whiskers” is meant elongated needle-like bodies capable of beingproduced from numerous substances as described in “The CondensedChemical Dictionary, Seventh Edition, Ed. Arthur & Elizabeth Rose,Reinhold Publishing Corp., New York (1966). The invention is not meantto be limited to the material from which the whiskers are made butinstead is meant to define a needle-like shaped structure wherein saidwhisker is smaller than the cell for which it is intended to be used inthe transformation thereof. It is within the scope of this invention forwhiskers to be shaped in a manner whereby DNA entry into a cell isfacilitated. It is also intended that the scope of said inventioninclude any material having a needle-like shape, said needle-like shapedmaterial being able to perforate a plant cell with or without cell wallsand thus facilitate DNA uptake and plant cell transformation. It is alsointended that the scope of this invention not include microinjectiontechniques, such as wherein a DNA molecule is inserted into a cell bypassing said DNA through an orifice intrinsic to a needle, said needlebeing first inserted into said cell. Preferably, whiskers are metal orceramic needle-like bodies, with those most preferred being made ofeither silicon carbide or silicon nitride and being 30×0.5 μm to 10×0.3μm in size.

By “whisker-mediated transformation” is meant the facilitation of DNAinsertion into plant cell aggregates and/or plant tissues by whiskersand expression of said DNA in either a transient or stable manner.

In producing plant cell lines, tissues of interest are asepticallyisolated and placed onto solid initiation medium whereby processesassociated with cell differentiation and specialization occurring inorganized plant cell tissues are disrupted, thus resulting in saidtissues becoming dedifferentiated. Typically, initiation medium issolidified by adding agar or the like because callus cannot be readilyinitiated in liquid medium. Media are typically based on the N6 salts ofChu et al., (1978, Proc. Symp. Plant Tissue Culture, Peking Press, p43-56) being supplemented with sucrose, vitamins, minerals, amino acids,and in some cases, synthetic hormones. However, callus tissues can alsoproliferate on media derived from the MS salts of Murashige and Skoog,(1962 Physiol. Plant. 15: 473-497). Cultures are generally maintained ina dark, sterile environment at about 28° C.

Typically, plant cell lines are preferably derived from tissues found injuvenile leaf basal regions, immature tassels, hypocotyl tissue, andcotylendonary nodes. For maize and rice, plant cell lines which producemeristematic tissue can be used, with those from zygotic embryo tissuebeing most preferred. Tissues most preferred for producing said plantcell lines are isolated from developing maize ears 10 to 14 days afterpollination and non-germinated rice seed. Hypocotyl andcotyledon-derived tissues from seedlings are most preferred forproduction of cotton plant cell lines.

After placing said tissues on solid medium, new meristems arise afterseveral days to a few weeks from either the scutellar region, in thecase of corn and rice, or from hypocotyl or cotyledonary tissue in thecase of cotton. These new meristems produce undifferentiatedparenchymatous cell aggregates without the structural ordercharacteristic of the tissue from which they are derived. Plant cellaggregates lack any recognizable overall structure and contain only alimited number of the many different kinds of specialized cell typesfound in intact, organized plant tissues. Said aggregates have beenclassified into non-embryogenic and embryogenic depending on theirregenerative capacity, mode of reproduction, and tissue morphology(Franz, 1988, Ph.D. Thesis, University of Wageningen, The Netherlands).

In corn and rice, non-embryogenic calli are comprised of soft, granular,translucent tissue consisting of elongated, vacuolated cells incapableof plant regeneration. Alternatively, embryogenic, monocotyledonouscalli, being capable of somatic embryogenesis and plant regeneration,exist in three distinct morphotypes: Type I; Type II; and Type III.

Type I callus consists of compact, nodular, slow-growing embryogeniccallus which proliferates as a mixture of complex tissues exhibitingshoot and/or scutellar-like structures (Phillips et al, 1988, In, Cornand Corn Improvement, pp 345-387). Said callus is characterized by ahigh degree of cellular differentiation, well developed vascularstructures and has been referred to as compact embryogenic callus (U.S.Pat. No. 5,641,664 to Plant Genetic Systems). Essentially allmonocotyledonous plants have tissue from which Type I callus can beproduced. Plant regeneration in Type I callus normally occurs eitherthrough organogenesis by elongation of meristems (Green and Phillips,1975, Crop Sci., 15:417-421) and/or through somatic embryogenesis from awell defined root-shoot axis (Vasil et al., 1984, Amer. J. Bot.71:158-161). The origin of regenerated shoots in Type I callus is notalways obvious and appears to take place via sub-epidermal meristemformation (Franz and Schel, 1991, Can. J. Bot. 69:26-33).

Type II callus, which is not as common as Type I, consists of soft,friable, embryogenic cells and can only be generated from certainmonocotyledonous genotypes (Phillips et al, 1988, In, Corn and CornImprovement, pp 345-387). It grows rapidly, contains little or novascular elements and can be described as friable, embryogenic callus(U.S. Pat. No. 5,641,664 to Plant Genetics Systems). A distinguishingfeature of Type II callus is that it contains numerous globular somaticembryos attached to suspensor-like structures on its surface throughwhich plant regeneration appears to progress in clearly identifiablestages (Franz, 1988, Ph.D. Thesis, University of Wageningen, TheNetherlands).

Type III callus has only been most recently described. Said callus isformed only very rarely, is easily dispersed in liquid and does not haveany distinct somatic embryos on its surface. It has also been describedas “friable, non-mucilaginous” callus (Shillito et al., 1989,Bio/Technology 7:581-587) and consists predominately of undifferentiatedtissues capable of regeneration via somatic embryogenesis. Type IIIcallus is considered the ideal tissue for transformation andregeneration because cells thereof easily disassociate and disperse, andthus, readily form suspension cultures. This type of callus is mostrare, distinct from Type II callus, and can only be produced by visuallyselecting and preferentially enriching for it at each sub-culturepassage of Type II cultures (WO94/28148; Zeneca).

In cotton, as with other dicotyledonous plants, callus types aretypically not defined as Type I, II, or III. Moreover, hypocotyl- andcotyledon-derived callus cultures from cotton have been classified intonon-embryogenic and embryogenic depending on morphology and regenerativecapacity (Shoemaker et al., 1986 Plant Cell Rep. 3:178-181).Non-embryogenic callus is comprised of a loose, friable mass of cellsthat does not exhibit a strong cytoplasm staining reaction and cannot beused to readily regenerate plants. However, embryogenic callus appearsas a tightly compact, dense cytoplasmic mass of cells capable of plantregeneration via somatic embryogenesis. Somatic embryos producedtherefrom first appear as globular structures which gradually elongateand then begin to exhibit cotyledonary development.

Of the callus types disclosed herein, Type I and Type II callus culturesderived from monocotyledonous plants are preferred in the production andregeneration of plants. Transgenic maize plants generated viawhisker-mediated transformation are most preferably made from eitherType I or Type II cultures; whereas, Type I callus cultures are mostpreferred in the production and regeneration of transgenic rice plants.In addition, cotyledonary node derived and hypocotyl-derived culturesare preferred in the production of transgenic cotton plants, withcultures produced from cotyledon tissue being most preferred.

Shoot tips of plants, including maize, rice, and cotton, contain apicalmeristems where organ primordia form from apical initial andsubepidermal cells (Steeves and Sussex, 1989, Patterns in PlantDevelopment, Cambridge University Press). Meristems can be isolated,placed onto shoot multiplication medium, and induced to produce multipleshoots from which plants can be regenerated (Zhong et al., 1992, Planta187:483-489). Meristems, either freshly isolated or precultured toinitiate shoot multiplication, can serve as recipient tissues forwhisker-mediated transformation as taught in the present invention.Shoot tips containing meristematic regions are preferably removed fromdeveloping embryos (Lowe et al., 1995, Bio/Technology 13:677-682) orgerminating seedlings (Gould et al., 1991, Plant Physiol. 95:426-434),transformed using whiskers with or without an osmotic pretreatment, andplaced onto shoot proliferation medium containing a selection agentprior to plant regeneration (Zhong et al., 1996, Plant Physiol.110:1097-1107).

The heterologous DNA used for transformation herein may be circular,linear, double-stranded or single-stranded. Generally, said DNA is arecombinant vector plasmid and contains coding regions therein whichserve to promote expression of the heterologous gene of interest as wellas provide a selectable marker whereby those tissues containing saidgene can be identified. Preferably, these recombinant vectors arecapable of stable integration into the plant genome where selection oftransformed plant lines is made possible by having said selectablemarker expression driven either by constitutive, tissue-specific, orinducible promoters included therein.

One variable present in a heterologous DNA is the choice of the chimericgene. Chimeric genes, either in the sense or antisense orientation, areexpressed in plant cells under control of a constitutive,tissue-specific, developmental, or inducible promoter and the like.Preference for a particular chimeric gene is at the discretion of theartisan; however, chimeric genes can be, but are not limited to, fromplants, animals, or bacteria and the like and can used to expressproteins either not found in a non-transformed cell or found in atransformed cell. Chimeric genes can be also used for, but are notlimited to, up-regulation or down-regulation of an endogenous gene ofinterest.

Another variable is the choice of a selectable marker. Preference for aparticular marker is at the discretion of the artisan, but any of thefollowing selectable markers may be used along with any other gene notlisted herein which could function as a selectable marker. Suchselectable markers include but are not limited to aminoglycosidephosphotransferase gene of transposon Tn5 (Aph II) which encodesresistance to the antibiotics kanamycin, neomycin and G418, as well asthose genes which encode for resistance or tolerance to glyphosate;hygromycin; methotrexate; phosphinothricin (bialophos); imidazolinones,sulfonylureas and triazolopyrimidine herbicides, such as chlorsulfuron;bromoxynil, dalapon and the like.

In addition to a selectable marker, it may be desirous to use a reportergene. In some instances a reporter gene may be used with or without aselectable marker. Reporter genes are genes which are typically notpresent in the recipient organism or tissue and typically encode forproteins resulting in some phenotypic change or enzymatic property.Examples of such genes are provided in K. Weising et al. Ann. Rev.Genetics, 22, 421 (1988), which is incorporated herein by reference.Preferred reporter genes include the beta-glucuronidase (GUS) of theuidA locus of E. coli, the chloramphenicol acetyl transferase gene fromTn9 of E. coli, the green fluorescent protein from the bioluminescentjellyfish Aequorea victoria, and the luciferase genes from fireflyPhotinus pyrais. An assay for detecting reporter gene expression maythen be performed at a suitable time after said gene has been introducedinto recipient cells. A preferred such assay entails the use of the geneencoding beta-glucuronidase (GUS) of the uidA locus of E. coli asdescribed by Jefferson et al., (1987 Biochem. Soc. Trans. 15, 17-19) toidentify transformed cells.

Another variable is a promoter regulatory element. In addition to plantpromoter regulatory elements, promoter regulatory elements from avariety of sources can be used efficiently in plant cells to expressheterologous genes. For example, promoter regulatory elements ofbacterial origin, such as the octopine synthase promoter, the nopalinesynthase promoter, the mannopine synthase promoter; promoters of viralorigin, such as the cauliflower mosaic virus (35S and 19S), 35T (whichis a re-engineered 35S promoter, see PCT/US96/1682; WO 97/13402published April 17, 1997) and the like may be used. Plant promoterregulatory elements include but are not limited toribulose-1,6-bisphosphate (RUBP) carboxylase small subunit (ssu),beta-conglycinin promoter, phaseolin promoter, ADH promoter, heat-shockpromoters and tissue specific promoters.

Other elements such as matrix attachment regions, scaffold attachmentregions, introns, enhancers, polyadenylation sequences and the like maybe present and thus may improve the transcription efficiency or DNAintegration. Such elements may or may not be necessary for DNA function,although they can provide better expression or functioning of the DNA byaffecting transcription, stability of the mRNA and the like. Suchelements may be included in the DNA as desired to obtain optimalperformance of the transformed DNA in the plant. Typical elementsinclude but are not limited to Adh-intron 1, the alfalfa mosaic viruscoat protein leader sequence, the maize streak virus coat protein leadersequence, as well as others available to a skilled artisan.

Constitutive promoter regulatory elements may also be used therebydirecting continuous gene expression in all cells types and at all times(e.g., actin, ubiquitin, CaMV 35S, and the like). Tissue specificpromoter regulatory elements are responsible for gene expression inspecific cell or tissue types, such as the leaves or seeds (e.g., zein,oleosin, napin, ACP, globulin and the like) and may also be used.

Promoter regulatory elements may also be active during a certain stageof the plants' development as well as active in specific plant tissuesand organs. Examples of such include but are not limited topollen-specific, embryo specific, corn silk specific, cotton fiberspecific, root specific, seed endosperm specific promoter regulatoryelements and the like. Under certain circumstances it may be desirableto use an inducible promoter regulatory element responsible forexpression of genes in response to a specific signal, such as: physicalstimulus (heat shock genes); light (RUBP carboxylase); hormone (Em);metabolites; and stress. Other desirable transcription and translationelements functional in plants may also be used. Numerous plant-specificgene transfer vectors are known and available to the skilled artisan.

Heterologous DNA can be introduced into regenerable plant cell culturesvia whiskers-mediated transformation. While a general description of theprocess can be found in U.S. Pat. Nos. 5,302,523 and 5,464,765, both toZeneca, no protocols have been published to date for whisker-mediatedtransformation of Type I, Type II, hypocotyl or cotyledon regenerableplant cell lines.

In whisker-mediated transformation, DNA uptake into plant material isfacilitated by very small, elongated, needle-like particles comprised ofa biologically inert material. When said particles are agitated in thepresence of DNA and plant cell lines, one or more of the particlesproduce small punctures in the regenerable plant cell aggregates therebyallowing said aggregates to uptake the DNA. Cells which have taken upthe DNA are considered to be transformed. Some transformed cells stablyretain the introduced DNA and express it.

The elongated needle-like particles used in plant cell transformationare termed “whiskers” and are preferably made of a high density materialsuch as silicon carbide or silicon nitride; however, any material havinga needle-like structure wherein the size of said structure is smallerthan the cell intended to be transformed is within the scope of theinvention. More preferably, whiskers are made of silicon carbide and areeither Silar SC-9 or Alfa Aesar as described herein.

Before transformation, plant cell lines are preferably placed ontoanosmotic medium and allowed to incubate thereby enhancingtransformation over non-osmotically treated cells. Most preferred is anosmotic medium having therein about 36.4 g/L sorbitol and about 36.4 g/Lmannitol. These tissue are maintained on said medium for about 4 hbefore whisker-mediated transformation. Incubation on medium withosmoticum after transformation at the discretion of the artisan.However, transformation of non-osmotically treated plant cellsaggregates and tissues is also within the scope of this invention.

Callus cultures used herein for generation of transgenic plants shouldgenerally be about 3 weeks to about 20 weeks old depending on theculture type. Typically, callus should be about midway between transferperiods and thus beyond any lag phase that might be associated withtransfer to a new media or before reaching any stationary phasetypically associated with a culture being on a plate for an extendedperiod of time. However, plant material can be taken before or aftersub-culturing; therefore, harvest timing is not generally believed to becritical to practicing the invention as disclosed herein. The amount ofcallus tissue used in each transformation can vary with amounts of about100 mg to about 500 mg preferred, about 100 mg to about 250 mg beingmore preferred and about 200 mg tissue being most preferred.

For transformation, whiskers are typically placed in a small container,such as a conical or microfuge tube and the like, wherein is placed amixture comprising the DNA construct of interest, a liquid medium, andcallus tissue. The order in which materials are added is not significantto practicing the invention as disclosed herein. Thereafter, thecontainer is sealed and agitated. Unlike particles used in biolistictransformation of plant tissue (Sanford et al., 1990 Physiol. Plantarum,79:206-209; and U.S. Pat. No. 5,100,712), whiskers do not require anyspecial pretreatment with DNA carriers or precipitants prior to use suchas CaCl₂, spermidine, sheared salmon sperm DNA and the like.

Agitation time used in the transformation process can vary and istypically from between about 10 sec to about 160 sec. The amount ofwhiskers added per transformation can also vary from between about 1 mgto about 4 mg per tube. An inverse relationship is observed between theamount of whiskers added and the agitation time needed to obtain optimaltransformation. Therefore, the amount of whiskers added and theagitation time needed to achieve transformation is determinable by onehaving skill in the art. In addition, the volume of liquid medium addedcan vary from about 200 μL to about 1000 μL, with about 200 μL beingpreferred. Moreover, the amount of heterologous DNA added can vary froma preferred amount of about 10 μL to about 100 μL of 1 mg/mL solution.The volume of DNA added is not as critical of factor to the invention asdisclosed herein as the final DNA concentration. However, preferredfinal DNA concentrations are from about 0.03 μg/μL to about 0.14 μg/μL.The scope of the present invention is not intended to be limited to saidcontainer size, the amount or concentration of heterologous DNA added,the volume of heterologous DNA added, the amount of the liquid mediumadded, the amount of callus material added or the amount of whiskersadded as disclosed herein. The scope of the invention is also notintended to be limited by the instrumentation used to agitate themixture or whether agitation is accomplished by manual or mechanicalmeans.

Once the plant cell lines have been perforated and the heterologous DNAhas entered therein, it is necessary to identify, propagate, and selectthose cells which not only contain the heterologous DNA of interest butare also capable of regeneration. Said cells and plants regeneratedtherefrom can be screened for the presence or absence of theheterologous DNA by various standard methods including but not limitedto assessment of reporter gene expression. Alternatively, transmissionof a selectable marker gene along with or as part of the heterologousDNA allows those cells containing said DNA to be identified by use of aselective agent.

Selection of only those cells containing and expressing the heterologousDNA of interest is a critical step in production of fertile, transgenicplants. Selection conditions must be chosen in such a manner as to allowgrowth of transformed cells while inhibiting growth of untransformedcells, which initially, are far more abundant. In addition, selectionconditions must not be so severe as to cause transformed cells to losetheir plant regenerability, future viability or fertility. A skilledartisan can easily determine appropriate conditions for selectingtransformed cells expressing a particular selectable marker byperforming growth inhibition curves. Growth inhibition curves aregenerated by plotting cell growth versus selective agent concentration.Typically, selective agent concentrations are set at a concentrationwhereby almost all non-transformed cells are growth inhibited but yetare not killed. Preferred are selective agent concentrations wherein90-99% of non-transformed cells are growth inhibited but yet not killed.Most preferred are selective agent concentrations wherein 97-99% ofnon-transformed cells are growth inhibited but yet not killed.

Transformed callus tissues transferred and exposed to selective agentsare generally incubated on solid medium supportive of growth. The mediumpreferred for each type of tissue has been well defined in the art.After initial exposure to selective agents, tissues are transferredperiodically to fresh medium while maintaining selective agentconcentrations. After transformed cell mass has essentially doubled insize, masses showing the most growth and appearing to be healthy areselected and transferred to fresh medium having selective agentconcentrations wherein non-transformed cells will be killed. Repeatedselection and transference of growing cells to fresh medium resulteventually in a selected group of cells comprised almost exclusively oftransformed cells containing the heterologous DNA of interest.

Regeneration, while important to the present invention, may be performedin any conventional manner available to the skilled artisan. If cellshave been transformed with selectable marker gene, the selective agentmay be incorporated into the regeneration media to further confirm thatthe regenerated plantlets are transformed. After subsequent weeks ofculturing, regenerated plantlet immune to the selective agent can betransferred to soil and grown to maturity.

Callus and plant derived therefrom can be identified as transformants byphenotypic and/or genotypic analysis. For example, if an enzyme orprotein is encoded by the heterologous DNA, enzymatic or immunologicalassays specific for the particular enzyme or protein can be used. Othergene products may be assayed by using suitable bioassays or chemicalassays. Other techniques include analyzing the genomic component of theplant using methods as described by Southern ((1975) J. Mol. Biol.,98:503-517), polymerase chain reaction (PCR) and the like.

Plants regenerated from transformed callus are referred to as the ROgeneration or RO plants. Seed produced by various sexual crosses fromplants of this generation are referred to as R1 progeny. R1 seed arethen germinated to produce R1 plants. Successful transmission andinheritance of heterologous DNA to R1 plants and beyond should beconfirmed using the methods described herein.

Generally, the commercial value of transformed corn and progeny thereofwill be of greatest value if the heterologous DNA can be incorporatedinto many different hybrids. This may be achieved by incorporating theheterologous DNA into a large number of parental lines directly asdescribed herein by creating plant cell aggregates of said lines andtransforming said lines with whiskers. In addition, this may also beaccomplished by crossing initial transgenic fertile plants to normalelite inbred lines and then crossing the progeny thereof back to thenormal parent. Progeny from this cross will segregate such that someplants will contain the heterologous DNA of interest and some plantswill not. Crossing of lines is continued until the original normalparent has been converted to a genetically modified line containing theheterologous DNA of interest and also possessing essentially allattributes associated with that line originally. Corn breedingtechniques needed to accomplish elite germplasm lines and inbredsthereof are well known to the skilled artisan.

Particular embodiments of this invention are further exemplified in theExamples. However, those skilled in the art will readily appreciate thatthe specific experiments detailed are only illustrative of the inventionas described more fully in the claims which follow thereafter.

EXAMPLE 1 Establishment of Type I and Type II Maize Callus Tissue

For Type I callus initiation, zygotic embryos (1.5-3.0 mm) of thegenotypes H99, Pa91, and a proprietary line, 139/39 (U.S. Pat. No.5,306,864), were aseptically isolated, infra., and placed onto Type Imedium consisting of N6 macro-nutrients and vitamins (Chu, 1978, Proc.Symp. Plant Tissue Culture, pp. 43-56), B5 micro-nutrients (Gamborg etal., Exp. Cell Res. 50:151-158), 30 g/L sucrose, 2.9 g/L L-proline, 100mg/L myo-inositol, 100 mg/L casein hydrolysate, 37 mg/L sodium salt ofFerrous-ethylenediaaminetetraacetic acid (NaFeEDTA), 8 mg/L dicamba, 0.8mg/L 2,4-D, 4.9 mg/L AgNO₃ and 2.5 g/L GELRITE. Type I callus, initiatedfrom the scutellar region of the original zygotic embryo, consisted ofcompact embryogenic tissue exhibiting scutellar-like structures andshoot meristems. After several passages of selective sub-culturing at21-28 day intervals, the cultures were used for transformationexperiments described herein.

For Type II callus initiation, plants of a hybrid made by inter-matingtwo S₃ lines derived from a B73×A188 cross (Armstrong et al., 1991,Maize Genet. Coop. News Lett., 65:92-93) were grown under standardgreenhouse conditions and self- or sib-pollinated (Petolino andGenovesi, 1994, The Maize Handbook, pp. 701-704). Ten to 14 days afterpollination (DAP), developing ears were removed and surface sterilizedwith 70% (v/v) ethanol for 2 min followed by soaking in 20% (v/v)commercial bleach for 0.5 h containing a few drops of LIQUI-NOX, thenrinsed several times with sterile H₂O. Immature embryos (1.5-3.0 mm)were then isolated and placed onto initiation medium [N6 basal salts andvitamins (Chu, 1978, Proc. Symp. Plant Tissue Culture, pp. 43-56), 20g/L sucrose, 2.9 g/L L-proline, 100 mg/L enzymatic casein hydrolysate(ECH), 37 mg/L Fe-EDTA, 10 mg/L AgNO₃, 1 mg/L 2,4-dichloro-phenoxyaceticacid (2,4-D), and 2.5 g/L GELRITE (Schweizerhall, South Plainfield,N.J.) at pH 5.8]. After 2-3 weeks in the dark at 28° C., Type II calluswith numerous globular and elongated somatic embryos was obtained. Anadditional 2-3 selective subcultures were performed to enrich Type IIcultures with desirable morphology as described in Welter et al., 1995,Plant Cell Rep. 14:725-729. Once Type II morphology was established (4-6weeks), callus was transferred to maintenance medium (initiation mediumcontaining 6 mM L-proline and no AgNO₃). Type II callus was used fortransformation after about 16-20 weeks from culture initiation.

EXAMPLE 2 Construction of the Plasmid pUGN81-3 and pDAB418

The maize expression vector, pUGN81-3, containing the ubiquitin promoterregulatory element driving the β-glucuronidase gene was used asdisclosed herein. Plasmid pUGN81-3 was a 8730 base pairs double strandedplant transformation vector composed of the following sequences inclockwise order. Nucleotides 1 to 17 encoded a polylinker. Nucleotides18 to 2003 of pUGN81-3 were the maize ubiquitin promoter and firstintron thereof and were PCR amplified from genomic DNA of maize genotypeB73 (Christensen et al., (1992) Plant Mol. Biol. 18:675-689).Nucleotides 2004 to 2022 of pUGN81-3 encoded a polylinker Nucleotides2023 to 4154 of pUGN81-3 corresponded to nucleotides 2551 to 4682 ofplasmid pBI101 (Clontech, Palo Alto, Calif.) followed by a polylinkerwhich corresponded to bases 4155 to 4197 of pUGN81-3. Nucleotides4265-4776 of pUGN81-3 comprised the double-enhanced 35S promoter, withnucleotides 4265 to 4516 corresponding to nucleotides 7093 to 7344 ofthe Cauliflower Mosaic Virus genome (Franck et al., (1980) Cell21:285-294). Nucleotides 4525 to 4776 of pUGN81-3 were a duplication ofnucleotides 4265 to 4516 with linker comprising nucleotides 4517 to 4524between the duplicated sequence. Nucleotides 4777 to 4871 of pUGN81-3corresponded to bases 7345 through 7439 of the Cauliflower Mosaic Virusgenome (Franck et al., (1980) Cell 21:285-294). Nucleotides 4872 to 4891comprised linker. Nucleotides 4892 to 5001 of pUGN81-3 corresponded tonucleotides 167 to 277 of the Maize Streak Virus genome with base 187absent (Mullineaux et al., (1984) EMBO J. 3:3063-3068). Nucleotides 5002to 5223 corresponded to the modified first intron of the maize alcoholdehydrogenase gene (Adh1-S) (Dennis et al., (1984) Nucleic Acids Res.12:3983-4000). The modification resulted in removal of 343 nucleotides(bases 1313 to 1656) with bases 1222 to 1312 (intron 5′ end) andnucleotides 1657 to 1775 (intron 3′ end) of the maize Adh1-S generemaining. Nucleotides 5224 to 5257 of pUGN81-3 corresponded to MaizeStreak Virus (MSV) nucleotides 279 to 312. Both sections of the MaizeStreak Virus, hereinafter MSV, sequence comprised the untranslatedleader of the MSV coat protein V2 gene, and were interrupted in plasmidpUGN81-3 by the modified Adh1 intron. Nucleotides 5258 to 5814 ofplasmid pUGN81-3 corresponded to nucleotides 29 to 585 of thephosphinotricin acetyl transferase (BAR) gene of Streptomyceshygroscopicus (White et al., (1989) Nucleic Acids Res. 18:1062). Tofacilitate cloning, nucleotides 34 and 575 of the published sequencewere changed from A and G to G and A, respectively. This sequence servedas the selectable marker. Nucleotides 5815 to 5819 comprised linker.Nucleotides 5820 to 6089 of pUGN81-3 corresponded to nucleotides 4414 to4683 of plasmid pBI101 (Clontech, Palo Alto, Calif.) followed by linkersequence. The remaining sequence of pUGN81-3 (nucleotides 6095 to 8730)corresponded to the reverse complement of pUC19 (Yanish-Perron et al.,(1985) Gene 33:103-119).

The maize expression vector, pDAB418, containied the ubiquitin promoterregulatory element driving the β-glucuronidase gene was used someexpression studies. In addition this plasmid carried a second gene whichserved as a plant selectable marker. Plasmid pDAB418 was a 10,149 basepairs double stranded plant transformation vector composed of thefollowing sequences in clockwise order. Nucleotides 32 to 2023 ofpDAB418 were the maize ubiquitin (Ubi1) promoter and first intron, andwere PCR amplified from genomic DNA of maize genotype B73 (Christensenet al., (1992) Plant Mol. Biol. 18:675-689). Nucleotides 2024 to 2042 ofpDAB418 comprised linker sequence. Nucleotides 2043 to 3894 of pDAB418corresponded to nucleotides 2551 to 4402 of plasmid pBI101 (Clontech,Palo Alto, Calif.) followed by linker sequence (bases 3895 to 3904).Nucleotides 3905 to 4174 OF pDAB418 corresponded to 4414 to 4683 ofpBI101. Nucleotides 4175 to 4192 were composed of linker sequence. Base4193 through 6184 was composed of a second copy of the maize ubiquitinpromoter and first intron as describe above. This sequence was followedby linker sequence TA (6185 to 6196). Nucleotides 6197 to 6753 ofplasmid pDAB418 corresponded to nucleotides 29 to 585 of thephosphinotricin acetyl transferase (BAR) gene of Streptomyceshygroscopicus (White et al., (1989) Nucleic Acids Res. 18:1062). Tofacilitate cloning, nucleotides 34 and 575 of the published sequencewere changed from A and G to G to A, respectively. This sequence servedas the selectable marker and was regulated by the maize ubiquitinpromoter. Nucleotides 6754 to 6758 were composed of linker. Nucleotides6759 through 7472 functioned as the 3′ polyadenylation sequence andincludes bases 21728 through 22441 from the Agrobacterium tumefaciensoctopine Ti plasmid pTi15955 (Barker et al. (1983) Plant Mol. Biol. 2,335-350. Sequence 7473 through 7504 was composed of polylinker. Theremaining sequence of pDAB418 (nucleotides 7505 to 10149) correspondedto the reverse complement of nucleotides from the plasmid backbonederived from pUC19 (Yanish-Perron et al., (1985) Gene 33:103-119).

EXAMPLE 3 Whiskers Preparation, Optimization, and Type I Corn CallusTransformation

Type I callus was produced from different inbred lines. For testing,about 250 mg samples of each Type 1 callus was placed into 2.0 mLmicrofuge tubes (Brinkman Instuments, Inc., Westbury, N.Y.), 300 mL ofType I medium consisting of N6 macro-nutrients and vitamins (Chu, 1978,Proc. Symp. Plant Tissue Culture, pp. 43-56), B5 micronutrients (Gamborget al., Exp. Ce. Res. 50:151-158), 30 g/L sucrose, 2.9 g/L L-proline,100 mg/L myo-inositol, 100 mg/L casein hydrolysate, 37 mg/L NaFeEDTA, 8mg/L dicamba, 0.8 mg/L 2,4-D, 4.9 mg/L AgNO₃], 20 μL of DNA solution[1.0 mg/mL pDAB418 in 10 mM Tris, 1 mM EDTA, pH 8.0], and 200 μL of a 40μg/μL (SC-9) whisker suspension was added. Each tube was then agitatedfor 20 sec using a Vortex-Genie 2™ and the callus was transferred backto Type I medium solidified with 2.5 g/L GELRITE and allowed to incubatefor 16 h. Afterwards, callus was assayed for GUS expression asdescribed, infra., with results summarized in Table 1.

TABLE 1 GUS expression in Type I callus. GUS Expression Genotype Unitsper Sample H99 31 ± 26 Pa91 22 ± 8  139/39 3 ± 3

Transient GUS expression in Type I callus following whisker treatmentwas observed in all genotypes tested.

EXAMPLE 4 Whiskers Preparation, Optimization, and Type II Corn CallusTransformation

A sterilized suspension of silicon carbide whiskers was prepared bytaking about 40 mg of dry whiskers (Silar SC-9 from Advanced CompositeMaterials Corp., Greer, S.C.; Alfa Aesar from Johnson-Matthey, WardHill, Mass.) and placing them into a pre-weighed 2.0 mL polypropylenetube. The tube was then re-weighed to determine the amount of whiskersadded, followed by autoclaving. Immediately before use, a 40 mg/mLwhisker suspension was made by adding maintenance medium, supra., andvortexing at high speed for 1 min.

In the transformation experiments, either 200 or 500 mg samples of TypeII callus from different immature embryo-derived lines were placed into17×100 mm culture tubes (Falcon 2059, Becton Dickinson, Lincoln Park,N.J.). Into each was added 500 μL of liquid maintenance medium [minusGELRITE], 80 μL of DNA solution [0.5 μg/μL pDAB418 in 10 mM Tris, 1 mMEDTA, pH 8.0], and 50 μL of a 40 mg/mL (SC-9) whisker suspension. Eachtube was then agitated for 20 sec using a Vortex-Genie 2™ (ScientificIndustries, Bohemia, N.Y.). Negative control experiments were alsoperformed. In one case, callus tissue was treated with whiskers in theabsence of DNA. In the other cases, callus tissue was treated with DNAin the absence of whiskers. Regardless of the treatment, callus was thentransferred back to solid maintenance medium having 2.5 g/L GELRITE andallowed to incubate for 16 h in the dark at 28° C. Afterwards, calluswas placed into GUS assay solution [0.2 M sodium phosphate pH 8.0, 0.1mM each of potassium ferricyanide and potassium ferrocyanide, 1.0 Msodium EDTA, 0.5 mg/mL 5-bromo-4-chloro-3-indolyl-β-D-glucuronide, and0.6% v/v Triton x-100]. GUS expression, defined as counts of bluesectors per sample, was measured after 48 h of incubation at 37° C. inthe dark and is summarized in Table 2.

TABLE 2 GUS expression following whisker-mediated transformation ofcallus at a fixed DNA concentration (0.0635 μg/μL Final concentration)Callus Amount/tube GUS Expression Sample Units Per 200 mg callus +whiskers + DNA 79 ± 22 200 mg callus + whiskers − DNA 0 200 mg callus −whiskers + DNA 0 500 mg callus + whiskers + DNA 51 ± 20 500 mg callus +whiskers − DNA 0 500 mg callus − whiskers + DNA 0

GUS expression was only observed when whiskers and DNA were bothagitated in the presence of callus. Slightly higher transient GUSexpression was observed when 200 mg of callus per tube was used ascompared to 500 mg per tube.

To test the effect of different DNA concentrations, about 200 mg samplesof Type II callus from different immature embryo-derived lines wereplaced into a 17×100 mm culture tube (Falcon 2059, Becton Dickinson,Lincoln Park, N.J.). Into each was added 250 μL of liquid maintenancemedium, either 10, 25, or 50 μL of DNA (1.0 μL of /μL pUGN81-3 in 10 mMTris, 1 mM EDTA, pH 8.0), and 50 μL of a 40 mg/mL (SC-9) whiskersuspension. Each tube was then agitated for 20 sec using a Vortex-Genie2™ (Scientific Industries, Bohemia, N.Y.). The callus was thentransferred back to semi-solid maintenance medium and allowed toincubate for 16 h in the dark at 28° C. Afterwards, callus was assayedfor GUS expression as previously described with the results shown inTable 3.

TABLE 3 GUS expression following whisker-mediated transformation of 200mg callus at varied DNA concentrations. Final DNA Concentration GUSExpression Sample Units Per 0.032 μg/μL 105 ± 73 0.077 μg/μL  86 ± 570.143 μg/μL  96 ± 76

As little as 10 μg of DNA per tube (0.032 μg/μL) containing about 200 mgof callus resulted in transient GUS expression. Increasing the amount ofDNA did not appear to significantly increase transient GUS expression.

About 500 mg samples of Type II callus representing different immatureembryo-derived lines were prepared as described herein and placed into17×100 mm culture tubes (Falcon 2059, Becton Dickinson, Lincoln Park,N.J.). Into each was added 500 μL of liquid maintenance medium and 50 μLof a DNA solution [0.5 μg/μL pDAB418 in 10 mM Tris, 1 mM EDTA, pH 8.0].The callus/DNA mixture was then allowed to incubate for 1 h either atroom temperature or on ice. Callus was incubated in the absence of DNAas a negative control. Afterwards, 50 μL of a 40 μg/μL (SC-9) whiskersuspension was added and vortexed as described herein. The callus wasthen transferred back to maintenance medium solidified with 2.5 g/LGELRITE and allowed to incubate for 16 h in the dark at 28° C.Afterwards, the callus was assayed for GUS expression as describedpreviously. Results are summarized in Table 4.

TABLE 4 GUS expression following whisker-mediated transformation ofcallus pre-incubated with DNA for 1 h. DNA Incubation GUS ExpressionSample Units Per DNA + callus + whiskers 70 ± 65 DNA + callus → 1 h(ice)→ whiskers 23 ± 37 DNA + callus → 1 h(RT) → whiskers 60 ± 27

Incubation of callus with DNA before whisker agitation resulted in lowertransient GUS expression, especially when the incubation was at roomtemperature. Best results were observed when DNA was added immediatelyprior to whisker treatment.

About 200 mg samples of Type II callus representing different immatureembryo-derived lines were either placed onto osmotic medium [maintenancemedium containing 36.4 g/L sorbitol and 36.4 g/L mannitol] and allowedto incubate for 4 h or maintained on maintenance medium. The callus wasthen placed into 17×100 mm culture tubes (Falcon 2059, Becton Dickinson,Lincoln Park, N.J.) and into each was added 200 μL of either liquidosmotic medium or liquid maintenance medium, 20 μL of DNA solution [1.0μg/μL pUGN81-3 in 10 mM Tris, 1 mM EDTA, pH 8.0], and 50 μL of 40 μg/μL(Alfa) whisker suspension. Each tube was then agitated for 20 sec usinga Vortex-Genie 2™, callus was transferred back to either osmotic mediumor maintenance medium solidified with 2.5 g/L GELRITE and allowed toincubate for 16 h in the dark at 28° C. Callus was then tested for GUSexpression as described previously. Results are summarized in Table 5.

TABLE 5 GUS expression of callus tissue having been osmotically treatedbefore and/or after whisker-mediated transformation. GUS ExpressionOsmotic Treatment Units Per Sample None  72 ± 19 Before Only 245 ± 80After Only  67 ± 37 Before and After 222 ± 83

Transient GUS expression was highest in those cultures which included anosmotic treatment before whisker agitation.

About 200 mg samples of Type II callus representing different cell lineswere placed onto osmotic medium and allowed to incubate for 4 h. Thecallus was then placed into 17×100 mm culture tubes (Falcon 2059, BectonDickinson, Lincoln Park, N.J.) and into each was added 200 μL of liquidosmotic medium [minus GELRITE], 20 μL of DNA solution [1.0 mg/mLpUGN81-3 in 10 mM Tris, 1 mM EDTA, pH 8.0] and either 25, 50, or 100 μLof a 40 μg/μl (Alfa) whisker suspension. Each tube was then agitated foreither 20- or 60 sec using a Vortex-Genie 2™. The callus was thentransferred back to liquid osmotic medium, allowed to incubate for 16 hin the dark at 28° C., and then assayed for GUS expression as describedpreviously. Results are summarized in Table 6.

TABLE 6 GUS expression of callus tissue transformed with various amountsof whiskers for 20- and 60 sec. Whisker GUS Expression Sample Units PerAmount 20 seconds 60 seconds  25 μL (1 mg/tube)  61 ± 36 136 ± 80   50μL (2 mg/tube)  70 ± 46 183 ± 126 100 μL (4 mg/tube) 155 ± 92 175 ± 98 

Averaged over all whisker amounts, highest GUS expression was observedfollowing 60 sec agitation compared to 20 sec. However, a 60 secagitation appeared to substantially damage the callus. High GUSexpression was also observed when 100 μL of the whisker suspension wasused (4 mg whiskers/tube). Our data indicate that increased whiskeramounts can compensate for agitation time and visa versa. The mostpreferred conditions was 100 μL of whiskers vortexed for 20 sec.

About 500 mg samples of Type II callus representing different cell lineswere placed onto osmotic medium and allowed to incubate for 4 h. Thecallus was then placed into 17×100 mm culture tubes (Falcon 2059, BectonDickinson, Lincoln Park, N.J.) and into each was added 1000 μL of liquidosmotic medium, 27.7 μL of DNA solution [0.72 μg/μL pUGN81-3 in 10 mMTris, 1 mM EDTA, pH 8.0], and 200 μL of 40 μg/μL whisker suspensionusing either Silar SC-9 (Advanced Composite, Greer, S.C.) or Alfa Aesar(Johnson Matthey, Ward Hill, Mass.) whiskers. Each tube was thenagitated for 20 sec using a Vortex-Genie 2™ and the callus wastransferred back to osmotic medium solidified with 2.5 g/L GELRITE andallowed to incubate for 16 h at 28° C. Afterwards, callus was assayedfor GUS expression as described previously with results summarized inTable 7.

TABLE 7 Comparison of whisker type on transformation. Whisker Type GUSExpression Sample Units per Silar SC-9 113 ± 48 Alfa Aesar 51 ± 9

Although both worked, better transient GUS expression was observed usingSilar SC-9 whiskers than Alfa Aesar whiskers.

About 500 mg samples of Type II callus representing different cell lineswere placed onto osmotic medium and allowed to incubate for 4 h. Thecallus was then placed into either 17×100 mm culture tubes (Falcon 2059,Becton Dickinson, Lincoln Park, N.J.) or 15 mL conical centrifuge tubes(Corning 25319-15, Corning, N.Y.). Into each was added 1000 μL of liquidosmotic medium, 20 μL of DNA solution [1.0 mg/mL pUGN81-3 in 10 mM Tris,1 mM EDTA, pH 8.0], and 200 μL of 40 μg/μL (Alfa) whisker suspension.Each tube was then agitated for 20 sec using either a Vortex-Genie or aCaulk Vari-Mix II (Estrada Dental, Rancho Cucamonga, Calif.). Callus wasthen transferred back to solid osmotic medium, allowed to incubate for16 h and then assayed for GUS activity (Table 8) as describedpreviously. High levels of transient GUS expression were observedfollowing agitation using both the Vortex and Vari-Mix; however,vortexing appeared to be somewhat better.

TABLE 8 The effect of vessel and agitator type on the transformation ofcallus tissue with whiskers. GUS Expression Vessel Units Per Sample TypeVortex Van-Mix Falcon 418 ± 202 256 ± 261 Corning 395 ± 362 353 ± 271Agitator Average 407 ± 294 304 ± 270

EXAMPLE 5 Production and Regeneration of Stably Trnsformed TransgenicMaize Plants

About 200 mg samples of Type II callus were placed onto osmotic mediumand allowed to incubate for 4 h. The callus was then placed into 17×100mm culture tubes (Falcon 2059, Becton Dickinson, Lincoln Park, N.J.) andinto each was added 200 μL of liquid osmotic medium, 20 μL of DNAsolution [1.0 μg/μL pUGN81-3 in 10 mM Tris, 1 mM EDTA, pH 8.0], and 100μL of 40 μg/μL (Alfa) whisker suspension. Each tube was then agitatedfor 20 sec using a Vortex-Genie 2™, the callus was transferred back toosmotic medium solidified with 2.5 g/L GELRITE and allowed to recoverfor ca. 48 h prior to selection.

The whisker-treated callus was placed onto selection medium [maintenancemedium with 30 mg/L Basta™ (Hoechst, Frankfurt, Germany) and noenzymatic casein hydrolysate or L-proline] and transferred to freshselection medium every four weeks for about 3-4 months. After 10-20weeks, actively growing colonies were isolated and sub-cultured ontofresh selection medium every two weeks to bulk-up callus prior toregeneration.

For plant regeneration, callus was transferred to induction medium,infra., and incubated at 280° C., 16h/8h light/dark photoperiod in lowlight (13 mE/m²/sec) for one week followed by 28° C., 16h/8h light darkphotoperiod in high light (40 mE/m²/sec) for one week provided by coolwhite fluorescent lamps. The induction medium was composed of MS saltsand vitamins (Murashige and Skoog, 1962, Physiol. Plant. 15:473-497), 30g/L sucrose, 100 mg/L myo-inositol, 5 mg/L benzyl amino purine, 0.025mg/L 2,4-D, 2.5 g/L GELRITE and adjusted to pH 5.7. Following thistwo-week induction period, callus was transferred to regeneration mediumand incubated in high light (40 mE/m²/sec) at 280° C. The regenerationmedium was composed of MS salts and vitamins, 30 g/L sucrose, and 2.5g/L GELRITE adjusted to pH 5.7. The callus was sub-cultured to freshregeneration medium about every two weeks until plantlets appeared. Bothinduction and regeneration medium contained 30 mg/L Basta™. Plantletswere transferred to 10 cm pots containing approximately 0.1 kg of dryMetro-Mix 360 (The Scotts Co, Marysville, Ohio), placed in a greenhouse,moistened thoroughly, and covered with clear plastic cups for 2-4 days.At the 3-5 leaf stage, plants were transplanted to 5-gallon potscontaining soil and grown to maturity.

EXAMPLE 6 Southern Analysis of Transformed Callus and Plant Tissues

BASTA resistant lines were characterized by Southern analysis to confirmthe presence of the transgene using a DNA probe specific for the codingregion of the gene of interest. DNA from both callus and leaf materialwas analyzed.

For callus, the material was soaked in distilled water for 30 min. andtransferred to a new petri dish prior to lyophilization. Leaf materialfrom plants was harvested at the 6-8 leaf stage. Genomic DNA wasprepared from lyophilized tissue as described by Saghai-Maroof et. al.((1984) Proceed. Nat. Acad. Sci. USA 81:8014-8018). Eight μg of each DNAwas digested with the restriction enzyme(s) specific for each plasmidconstruct using conditions suggested by the manufacturer (BethesdaResearch, Gaithersburg, Md.) and separated by electrophoresis on a 0.8%agarose gel. The DNA was then blotted onto nylon membranes as describedby Southern ((1975) J. Mol. Biol., 98:503-517). A 1.9 kb DNA probespecific for the GUS coding region was prepared using an OligolabellingKit (Pharmacia LKB, Piscataway, N.J.) with 50 mCi of [a-³²P]dCTP(Amersham Life Science, Arlington Heights, Ill.). The radioactive probewas then hybridized to the genomic DNA on the blots in 45 mL of minimalhybridization buffer [10% polyethylene glycol, 7% sodium dodecyl sulfate(SDS), 0.6×SSC where 1×SSC is 0.15 M NaCl, 0.015 M sodium citrate, pH7.0, 10 mM sodium phosphate, 5 mM EDTA and 100 μg/mL denatured salmonsperm DNA] overnight at 60° C. After hybridization, blots were washed at60° C. in 0.25×SSC and 0.2% SDS for 45 min., blotted dry and exposed toXAR-5 film (Kodak, Rochester, N.Y.) overnight on two intensifyingscreens (DuPont, Newark, Del.).

Plants regenerated from transgenic callus lines were tested via Southernanalysis and all were found to have the 4.2 kb hybridization product.The hybridizing product was expected to contain the ubiquitin-1promoter/intron, the GUS coding sequence, and the nos 3′ untranslatedregion. Individual plants regenerated from a given culture all displayedidentical hybridization patterns.

EXAMPLE 7 Establishment of Embryogenic Type I Rice Callus Tissue

For initiation of rice embryogenic Type I callus cultures, mature seedsof a Oryza sativa L. cv. Japonica, Taipei 309, were dehusked andsurface-sterilized in 70% (v/v) ethanol for 2-5 min followed by a 30-45min soak in 50% (v/v) commercial bleach containing a few drops ofLIQUI-NOX (Alconox, Inc., New York, N.Y.). The seeds were then rinsed 3times in sterile H₂O and placed on filter paper before being transferredto induction medium [N6 macro elements (Chu, 1978, Proc. Symp. PlantTissue Culture, Peking Press, p 43-56), B5 micro elements and vitamins(Gamborg et al., 1968, Exp. Cell Res. 50:151-158), 300 mg/L caseinhydrolysate, 500 mg/L L-proline, 500 mg/L L-glutamine, 30 g/L sucrose, 2mg/L 2,4-D, and 2.5 g/L GELRITE, pH 5.8]. The seeds were cultured oninduction medium and incubated in the dark at 28° C. for 3 weeks.Afterwards, emerging primary callus induced from the scutellar regionwas transferred to fresh induction medium for further maintenance. Ingeneral, embryonic callus was selected based on its morphology aftereach subculture. The general morphology of rice Type I embryogeniccallus emerged as hard, compact, nodular structures appearing, and insome cases not appearing, embryo-like. This material was termed Type I.

EXAMPLE 8 Whiskers Preparation, Optimization, and Rice Type I CallusTransformation

Immediately prior to DNA delivery, a sterile 50 μg/μL suspension ofsilicon carbide whiskers (Silar SC-9) was prepared in water as describedpreviously. About 125-500 mg of Type I rice embryogenic callus beingless than 4 months-old was selected and transferred into either a 15 mLCorning conical centrifuge tube or a 1.5 mL microfuge tube to whichabout 250 μL of liquid initiation medium (initiation medium withoutGELRITE) had been added. Then, about 10-160 μL of a 50 μg/μL whiskersolution was added along with 25 μL of 1 μg/μL pDAB418 DNA solutionimmediately prior to vortexing for 15-120 sec with Vortex Genie 2™ atthe highest speed possible. Afterwards, the callus was transferred toinitiation medium with and without high osmoticum [0.2 M mannitol and0.2 M sorbitol; Vain et al., 1993, Plant Cell Rep. 12:84-88] andincubated in the dark at 28° C. for about 24 h before being examined forGUS activity.

Gus histochemical assays were conducted according to Jefferson et al.,(1987, EMBO J 6: 3901-3907) and as described here. Tissues were placedin 24-well microtiter plates (Corning, New York, N.Y.) containing 500 μLof assay buffer per well. The assay buffer consisted of 0.2 M sodiumphosphate (pH 8.0), 0.1 mM potassium ferricyanide, 0.1 mM potassiumferrocyanide, 1.0 M sodium EDTA, 1.9 mM5-bromo-4-chloro-3-indolyl-β-D-glucuronide, and 0.06% triton X-100. Theplates were incubated in the dark for 1-2 days at 37° C. beforemicroscopically examining the tissue for expression. Silicon carbidewhisker-mediated DNA delivery was measured in terms of GUS transientexpression, i.e., GUS expression units (blue spots) per sample.

Many different transformation parameters were tested to determine theoptimum conditions needed for transformation. Some of the parameterstested included: vortexing time, amount of the 50 μg/μL whisker solutionadded, treatment with high osmoticum before and/or after whiskertreatment, amount of tissue used per transformation, and vessel size. Inall of these experiments, the protocols described herein wereessentially the same unless otherwise mentioned.

The effect of vortexing time (15-60 sec) was studied for DNA deliveryinto rice embryogenic Type I callus. In this experiment, callus wastreated with high osmoticum for about 4 h prior to transformation withwhiskers and for about 16 h following whisker treatment. Callus wastransformed by placing 250 mg of callus into a 15 mL Corning conicalcentrifuge tube, adding 1000 μL of liquid initiation medium, 25 μL of a1 μg/μL DNA solution (pDAB418), and 100 μL of a 50 μg/μL whiskersolution. Multiple sample sets were vortexed for 15-, 30-, 60- and 120sec and the results were quantitated as shown in Table 9.

TABLE 9 The effect of vortexing time on the transformation of rice TypeI embryogenic callus. Vortexing Gus Expression Time (Sec) Units perSample 15 113.5 30 107.5 60 90.5 120 72.5

As can be seen, DNA delivery and callus transformation was possible whenvortexing for as little as 15 sec despite the hard and compactmorphology of rice Type I embryogenic callus.

The effect of varying the amount of whiskers added was examined whilemaintaining a constant volume (1000 μL) of liquid initiation medium.Callus samples were treated with high osmoticum before and afterwhisker-mediated transformation. About 250 mg of callus tissue wasselected and transferred to a 15 mL Corning conical centrifuge tube towhich 1,000 μL of liquid initiation medium lacking GELRITE was added.Different amounts of a 50 μg/μL whisker solution (10, 20, 40, 80, and160 μL) along with 25 μL of a 1 μg/μL DNA solution (pDAB418) were addedprior to vortexing with Vortex Genie 2™ for 60 sec. Samples were thentransferred back onto the initiation medium with high osmoticumovernight before histochemical GUS assays were performed. The resultsare described in Table 10. GUS expression was found to correlate with anincreasing abundance of whiskers added.

TABLE 10 The effect of increasing the amount of whiskers whilemaintaining a constant volume of initiation medium. Whisker Solution GusExpression Added (μL) Units/Sample 10 2 20 1 40 9 80 6 160 6.5

The effect of high osmoticum on transformation efficiency was studiedwith the results summarized in Table. 11. In this experiment, callustissue was subjected to either no high osmoticum treatment or highosmoticum treatment after whisker-mediated transformation. About 250 mgof callus tissue was transferred to 1.5 mL micofuge tube. About 250 μLof liquid initiation medium was added followed by 125 μL of 50 μg/μLwhisker solution and 25 μL of a 1 μg/μL DNA solution (pDAB418). Thesamples were vortexed for 60 sec and transferred back onto theinitiation medium overnight with or without high osmoticum before GUSassays were conducted.

TABLE 11 The effect of osmoticum treatment after vortexing and vortexingtime on transformation and transgene expression of rice Type Iembryogenic callus tissue. GUS Expression Osmoticum After Vortex UnitsTransformation mg Tissue Sec. Per Sample + 250 30 48 + 250 60 44 + 50030 37 + 500 60 44 − 250 30 87 − 250 60 107 − 500 30 96 − 500 60 120

Callus tissue with no high osmoticum treatment after whiskertransformation resulted in higher expression levels compared to thatwhere high osmoticum treatment occurred.

The amount of callus tissue added and vessel size (1.5 mL microfuge tubevs. 2.0 mL microfuge tube) used were studied. In this experiment, 125 or250 mg of callus tissue were used while maintaining a constant volume ofliquid initiation medium, i.e., 250 μL. In all cases, 125 μg/μL of a 50μg/μL whisker solution along with 25 μL of a 1 μg/μL DNA solution(pDAB418) were added prior to vortexing with Vortex Genie 2™ for 60 sec.Following transformation, callus samples were incubated on theinitiation medium with high osmoticum overnight before GUS assays wereconducted. The results are summarized in Table 12.

TABLE 12 The effect of vessel size and the amount of tissue added on thewhiskers-mediated transformation of rice Type 1 callus tissue. mg TissueVessel Size (mL) Gus Expression Units/Sample 125 1.5 8 125 2.0 6 250 1.524 250 2.0 9.5

The use of 1.5 mL microfuge tube and 250 mg callus tissue per tuberesulted in highest transient expression.

EXAMPLE 9 Plasmid Description for pUbiHyg

The rice expression vector, pUbiHyg, contained the maize ubiquitinpromoter and first intron from the ubiquitin gene (Ubi1) regulatoryelement driving the hygromycin B phosphotransferase (resistance) gene asdescribed by Gritz and Davies, (1983) Gene 25:179-188. Plasmid pUbiHygwas a 5991 base pairs double stranded plant transformation vectorcomposed of the following sequences in clockwise order. Nucleotides 43through 2034 of pUbiHyg were the maize ubiquitin promoter and firstintron, and were PCR amplified from genomic DNA of maize genotype B73(Christensen et al., (1992) Plant Mol. Biol. 18:675-689) Nucleotides2053 through 3078 of pUbiHyg corresponded to nucleotides 211 through1236 of the the hygromycin B phosphotransferase (resistance) genesequence (accession number K01193), with bases 2056 and 2057 of pUbiHygmodified from AA to GT to facilitate future cloning. Bases 3079 through3097 were composed of linker. Bases 3098 through 3351 corresponded tonucleotides 4430 to 4683 of plasmid pBI101 (Clontech, Palo Alto, Calif.)The remaining sequence of pUbiHyg (nucleotides 3352 to 5991)corresponded to nucleotides from the plasmid backbone, (Yanish-Perron etal., (1985) Gene 33:103-119).

EXAMPLE 10 Production of Stably Transformed Rice Type I Callus Tissueand Regeneration of Transgenic Plants

For production of stable rice transgenic plants, rice Type I callustissue was produced as described herein and subjected towhisker-mediated transformation. Typically, 250 mg of rice Type Icallus, with or without high osmoticum treatment for four hours, wasadded to a 1.5 microfuge tube. Also added were 250 μL of liquidinitiation medium, 125 μL of a 50 μg/μL whisker solution, and 25 μL of a1 μg/μL DNA solution (pDAB 305 and UbiHyg in 1:1 ratio). Vortexing wascarried out for 60 sec as described previously to ensure DNA delivery.

Following whisker-mediated DNA delivery, callus was transferred toinitiation medium, as described previously, with high osmoticum for 24 hbefore transfer to selection medium. Selection medium consisted ofinitiation medium with 30 mg/L hygromycin (CalBiochem-NovabiochemCorporation, La Jolla, Calif.). After 2 weeks, cultures were transferredto fresh selection medium having 50 mg/L hygromycin (Li et al., 1993,Plant Cell Rep. 12: 250-255). Placing cultures on selection mediumresulted in the formation of compact, white-yellow, embryogenic calluscultures after 30-45 days. These cultures were then transferred topre-regeneration (PR) medium having 50 mg/L hygromycin and maintained inthe dark at 28° C. PR medium consisted of initiation medium with 2 mg/Lbenzyl aminopurine (BAP), 1 mg/L naphthalene acetic acid (NAA), and 5mg/L abscisic acid (ABA). After 2 weeks, cultures were then transferredto regeneration (RN) medium wherein RN medium was initiation medium with3 mg/L BAP, and 0.5 mg/L NAA. Cultures on RN medium were incubated forat 28° C. under high fluorescent lights (325-ft-candles) until plantletsemerged. When the plantlet shoots reached about 2 cm, they weretransferred to Magenta GA7 boxes (Magenta Corp., Chicago, Ill.)containing medium consisting of MS macro and micro nutrients along withB5 vitamins (Gamborg et al., 1968, Exp. Cell Res. 50:151-158) which hadbeen diluted 1:1 with water, 10 g/L sucrose, 0.05 mg/L NAA, 50 mg/Lhygromycin, 2.5 g/L GELRITE, and adjusted to pH 5.8. When plantlets wereestablished with well-developed root systems, they were transferred tosoil/metromix 360 (1:1) and raised in the greenhouse (29°/24° C.day/night cycle, 50-60% humidity, 12 h photoperiod) until maturity.Southern analysis of these plants revealed the presence of thehygromycin gene indicating that they were indeed transformed. Theseplants were successfully grown to maturity in the greenhouse.

EXAMPLE 11 Plasimd Description for pSMGN179-3

The expression vector, pSMGN179-3, contained a modified derivative ofthe chimeric regulatory regions from the Agrobacterium tumefaciens opinesynthase genes described by Gelvin and Hauptmann, (1995) Patent WO95/14098 driving the β-glucuronidase gene. Plasmid pSMGN179-3 was a 5541base pairs double stranded plant transformation vector composed of thefollowing sequences in clockwise order: nucleotides 1 to 16 had themultiple cloning sequence from the plasmid backbone, pUC19(Yanish-Perron et al., (1985) Gene 33:103-119). Nucleotides 17 to 57 ofpSMGN179-3 were composed of linker fragment. Bases 58 through 277 ofpSMGN179-3 corresponded to the reverse complement of bases 13774 through13993 of Agrobacterium tumefaciens Ti plasmid pTi15955 T-DNA (Barker etGal. (1983) Plant Mol. Biol. 2:335-350). These bases corresponded toupstream activating sequences from the octopine synthase (ocs) gene.Bases 278 through 304 corresponded to linker DNA. Bases 305 through 350corresponded to the reverse complement of bases 21475 through 21520 ofAgrobacterium tumefaciens Ti plasmid pTi15955 T-DNA (Barker et Gal.(1983) Plant Mol. Biol. 2:335-350). Bases 351 through 737 of pSMGN179-3were composed of the reverse complement of bases 20128 through 20514from Agrobacterium b-glucuronidase gene tumefaciens Ti plasmid pTi15955T-DNA (Barker et Gal. (1983) Plant Mol. Biol. 2:335-350). The sequenceof pSMGN179-3 from 305 through 737 included the ΔMAS promoter describedby Gelvin and Haupman, (1995) Patent WO 95/14098. Bases 738 through 763of pSMGN179-3 contain linker sequence. Bases 58 through 763 ofpSMGN179-3 comprised the promoter and untranslated leader regulatoryfusion as given in SEQ ID NO 1. Nucleotides 764 to 2615 of pSMGN179-3corresponded to nucleotides 2551 to 4402 of plasmid pBI101 which encodedthe β-glucuronidase gene (Clontech, Palo Alto, Calif.)(Jefferson et al.,(1986) Plant Mol. Biol. Rep. 83(22):8447-8451). Bases 767 through 769were modified from TTA to GTC. The β-glucuronidase gene was followed bypolylinker sequence, corresponding to bases 2616 to 2625 of pSMGN179-3.Bases 2626 through 2895 corresponded to 4414 to 4683 of pBI101 whichcontained the sequence from the nopaline synthase 3′ untranslatedregions (Clontech, Palo Alto, Calif.)(Jefferson et al., (1986) PlantMol. Biol. Rep. 83(22):8447-8451). The remaining sequence of pSMGN179-3(nucleotides 4684 to 5541) corresponded the reverse complement ofnucleotides from the plasmid backbone which was derived from pUC19(Yanish-Perron et al., (1985) Gene 33:103-119).

EXAMPLE 12 Transformation of Embryogenic Cotton Callus

Approximately 250 mg of cotton callus from different seedling-derivedlines were placed onto osmotic medium [maintenance medium plus 36.4 g/Lsorbitol and 36.4 g/L mannitol] and incubated for 4 h in the dark. Thecallus was then placed into a 17×100 mm culture tube (Falcon 2059,Becton Dickinson, Lincoln Park, N.J.) into which was added 250 μL ofliquid osmotic medium [minus GELRITE], 20 μL of DNA (1.0 μg/μLpSMGN179-3 in 10 mM Tris, 1 mM EDTA, pH 8.0), and 50 μL of a 40 mg/mL(Alfa) whisker suspension. Each tube was then agitated for either 40,80, or 160 sec using a Vortex-Genie 2™ (Scientific Industries, Bohemia,N.Y.). The callus was then transferred back to osmotic medium solidifiedwith 2.5 g/L GELRITE and allowed to incubate at 28° C. in the dark for16 h. Afterwards, it was assayed for GUS expression as previouslydescribed with the results shown in Table 13.

TABLE 13 GUS expression following whisker-mediated transformation cottoncallus using different agitation times. Agitation Time Sample GUSExpression Units Per  40 sec 41 ± 21  80 sec 59 ± 38 160 sec 37 ± 28

GUS expression was observed in whisker-treated cotton callus. Increasingthe agitation time from 40 to 160 sec did not appear to significantlyincrease expression.

EXAMPLE 13 Production of Plasmid pDAB 305

Plasmid pDAB305 was a 5800 bp plasmid that harbored a promotercontaining a tandem copy of the Cauliflower Mosaic Virus 35S enhancer(35S), a deleted version of the Adh1 intron 1, and the untranslatedleader from the Maize Streak Mosaic Virus Coat Protein fused to theβ-glucuronidase gene, which was then followed by the nos 3′ UTR. It wasmade as follows:

The starting material was plasmid pUC13/35S(-343) as described by Odellet al. (1985 Nature 313:810-812). This plasmid comprised, starting atthe 3′ end of the Sma I site of pUC13 (Messing, 1983 in Methods inEnyzmology, Wu, R. Ed. 101:20-78), and reading on the strand contiguousto the noncoding strand of the lacZ gene of pUC13, nucleotides 6495 to6972 of CaMV, followed by linker sequence which encodes a Cla Irecognition site), followed by CaMV nucleotides 7089 to 7443, followedby linker sequence, the latter sequence comprising the recognitionsequence for Hind III, which was then followed by the remainder of thepUC13 plasmid DNA.

The plasmid pUC13/35S(-343) DNA was digested with Cla I and Nco I, the3429 base pair (bp) large fragment was separated from the 66 bp smallfragment by agarose gel electrophoresis, and then purified by standardmethods. The DNA was digested with Cla I, and the protruding ends weremade flush by treatment with T4 DNA polymerase. The blunt-ended DNA wasthen ligated to synthetic oligonucleotide linkers, which included an NcoI recognition site. The ligation reaction was transformed into competentE. coli cells, and a transformant was identified that contained aplasmid (named pOO#1) that had an Nco I site positioned at the formerCla I site. DNA of pOO#1 was digested with Nco I and the compatible endsof the large fragment were religated, resulting in the deletion of 70 bpfrom pOO#1, to generate intermediate plasmid pOO#1 NcoΔ.

The plasmid pOO#1 NcoΔ DNA was digested with EcoR V, and the blunt endswere ligated to Cla I linkers having the sequence CATCGATG. An E. colitransformant harboring a plasmid having a new Cla I site at the positionof the previous EcoR V site was identified, and the plasmid was namedpOO#1 NcoΔ RV>Cla. This DNA was then digested with Cla I and Nco I, andthe small (268 bp) fragment was purified from an agarose gel. Thisfragment was then ligated to the 3429 bp Cla I/Nco I fragment ofpUC13/35S(-343) prepared above, and an E. coli transformant thatharbored a plasmid having Cla I/Nco I fragments 3429 and 268 bp wasidentified. This plasmid was named pUC13/35S En.

The plasmid pUC13/35S En DNA was digested with Nco I, and the protrudingends were made blunt by treatment with T4 DNA polymerase. The treatedDNA was then cut with Sma I, and was ligated to Bgl II linkers. An E.coli transformant that harbored a plasmid in which the 416 bp Sma I/NcoIfragment had been replaced with at least two copies of the Bgl IIlinkers was identified, and named p35S En². The DNA structure of p35SEn² was as follows: Beginning with the nucleotide that follows the thirdC residue of the Sma I site on the strand contiguous to the noncodingstrand of the lacZ gene of pUC13; linker sequence, followed by CaMVnucleotides 7090 to 7344, followed by the Cla I linker sequence,followed by CaMV nucleotides 7089 to 7443, followed by the Hind IIIlinker sequence, followed by the rest of pUC13 sequence. This structurehad the feature that the enhancer sequences of the CaMV 35S promoter,which laid in the region upstream of the EcoR V site in the viral genome(bases 7090 to 7344), had been duplicated. This promoter constructincorporated the native 35S transcription start site, which was 11nucleotides upstream of the first A residue of the Hind III site.

For plasmids utilizing the 35S promoter and the Agrobacterium NOS Poly Asequences, the starting material for the first construct was plasmidpBI221, purchased from CLONTECH (Palo Alto, Calif.). This plasmidcontained a slightly modified copy of the CaMV 35S promoter. Beginningat the 3′ end of the Pst I site of pUC19 (Yanisch-Perron et al., 1985Gene 33:103-119), and reading on the same strand as that which encodesthe lacZ gene of pUC19, the sequence was comprised of linkernucleotides, followed by CaMV nucleotides 6605 to 7439 followed bylinker sequence. These bases were then followed by 1809 bp comprisingthe coding sequence of the E. coli uidA gene, which encodes theβ-glucuronidase (GUS) protein, and 55 bp of 3′ flanking bases that arederived from the E. coli genome (Jefferson, 1986 Proc. Natl. Acad. Sci.83:8447-8451), followed by the Sac I linker sequence, which was thenfollowed by linker sequence. These bases were followed by the RNAtranscription termination/polyadenylation signal sequences derived fromthe Agrobacterium tumefaciens nopaline synthase (NOS) gene, andcomprised the 256 bp Sau3A I fragment corresponding to nucleotides 1298to 1554 of DePicker et al. (1982 J. Molec. Appl. Genet. 1:561-573),followed by two C residues, the Eco RI recognition sequence, and therest of pUC19.

The plasmid pBI221 DNA was digested with EcoR I and BamH I, and the 3507bp fragment was purified from an agarose gel. Plasmid pRAJ275 (CLONTECH)DNA was digested with EcoR I and Sal I, and the 1862 bp fragment waspurified from an agarose gel. These two fragments were mixed together,and complementary synthetic oligonucleotides were added. The fragmentswere ligated together, and an E. coli transformant harboring a plasmidhaving the appropriate DNA structure was identified by restrictionenzyme analysis. DNA of this plasmid, named pKA881, was digested withBal I and Eco RI, and the 4148 bp fragment was isolated from an agarosegel. DNA of pBI221 was similarly digested, and the 1517 bp Eco RI/Bal Ifragment was gel purified and ligated to the above pKA881 fragment, togenerate plasmid pKA882. Then pKA882 DNA was digested with Sac I, theprotruding ends were made blunt by treatment with T4 DNA polymerase, andthe fragment was ligated to synthetic BamH I linkers. An E. colitransformant that harbored a plasmid having BamH I fragments of 3784 and1885 bp was identified and named pKA882B. Following, pKA882B DNA wasdigested with BamH I, and the mixture of fragments was ligated. An E.coli transformant that harbored a plasmid that generated a single 3783bp fragment upon digestion with BamH I was identified and namedp35S/NOS. This plasmid had the essential DNA structure of pBI221, exceptthat the coding sequences of the GUS gene had been deleted. Therefore,CaMV nucleotides 6605 to 7439 were followed by linker sequence. Thelinker sequence was then followed by the NOS Polyadenylation sequencesand the rest of pBI221.

The plasmid p35S/NOS DNA was digested with EcoR V and Pst I, and the3037 bp fragment was purified and ligated to the 534 bp fragmentobtained from digestion of p35S En² DNA with EcoR V and Pst I. An E.coli transformant was identified that harbored a plasmid that generatedfragments of 3031 and 534 bp upon digestion with EcoR V and Pst I, andthe plasmid was named p35S En²/NOS. This plasmid contained theduplicated 35S promoter enhancer region described for p35S En², thepromoter sequences being separated from the NOS polyadenylationsequences by linker sequences that included unique Xba I and BamH Isites.

The MSV genomic sequence was published by Mullineaux et al., (1984 EMBOJ. 3:3063-3068), and Howell (1984 Nucleic Acids Res. 12:7459-7375), andthe transcript was described by Fenoll et al. (1988 EMBO J.7:1589-1596). The entire sequence, comprised 154 bp and was constructedin three stages by assembling blocks of synthetic oligonucleotides.First, complementary oligonucleotides were synthesized and purified bystandard procedures. Annealing of these nucleotides into double-strandedstructures left 4-base single stranded protruding ends compatible withthose generated by BamH I on one end of the molecule, and with HindIII-generated single stranded ends on the other end of the molecule.Such annealed molecules were ligated into plasmid pBluescript SK(−)[hereinafter called pBSK; Stratagene Cloning Systems, La Jolla, Calif.],that had been digested with BamH I and Hind III. The sequence of theseoligonucleotides was such that when ligated onto the respective BamH Iand Hind III sticky ends, the sequences of the respective recognitionsites were maintained. An E. coli transformant harboring a plasmidcontaining the oligonucleotide sequence was identified by restrictionenzyme analysis, and the plasmid was named pMSV A.

Complementary oligonucleotides were synthesized and purified by standardprocedures. Annealing of these nucleotides into double-strandedstructures left 4-base sticky ends that were compatible with thosegenerated by Hind III on one end of the molecule and with SalI-generated sticky ends on the other end of the molecule (TCGA). Thesequence of these oligonucleotides was such that when ligated onto theHind III sticky ends the recognition sequence for Hind III wasdestroyed.

DNA of pMSV A was digested with Hind III and Sal I, and was ligated tothe above annealed oligonucleotides. An E. coli transformant harboring aplasmid containing the new oligonucleotides was identified byrestricition enzyme site mapping, and was named pMSV AB.

Complementary oligonucleotides were synthesized and purified by standardprocedures. These oligonucleotides incorporate bases that compriserecognition sites for Nco I, EcoR V, Hind III, and BamH I. Annealing ofthese nucleotides into double-stranded structures left 4-base stickyends that were compatible with those generated by Xma I on one end ofthe molecule, and with Xho I-generated sticky ends on the other end ofthe molecule. Such annealed molecules were ligated into pMSV AB DNA thathad been digested with Xma I and Xho I. An E. coli transformantharboring a plasmid containing the oligonucleotide sequence wasidentified by restriction enzyme analysis, and DNA structure wasverified by sequence analysis. The plasmid was named pMSV CPL. Together,these comprised the 5′ untranslated leader sequence (“L”) of the MSVcoat protein (“CP”) gene. These corresponded to nucleotides 167 to 186,and 188 to 317 of the MSV sequence of Mullineaux et al., (1984), andwere flanked on the 5′ end by BamH I linker sequence, and on the 3′ endby linker sequence. An A residue corresponding to base 187 of the wildtype MSV sequence was inadvertently deleted during cloning.

The plasmid pMSV CPL DNA was digested at the Sma I site corresponding tobase 277 of the MSV genomic sequence, and the DNA was ligated to Bgl IIlinkers. An E. coli transformant harboring a plasmid having a unique BglII site at the position of the former Sma I site was identified andverified by DNA sequence analysis, and the plasmid was named pCPL-Bgl.

The starting material in construction of a deleted version of the maizealcohol dehydrogenase 1 (Adh1) intron 1 is plasmid pVW119, which wasobtained from V. Walbot, Stanford University, Stanford, Calif. Thisplasmid contained the DNA sequence of the maize Adh1.S gene, includingintron 1, from nucleotides 119 to 672 [numbering of Dennis et al. (1984Nucleic Acids Res. 12:3983-4000)], and was described in Callis et al.(1987 Genes and Develop. 1:1183-1200). The entire intron 1 sequence,with 14 bases of exon 1, and 9 bases of exon 2, was obtained from thisplasmid on a 556 bp fragment following digestion with Bcl I and BamH I.

The plasmid pSG3525a(Pst) DNA was digested with BamH I and Bcl I, andthe 3430 bp fragment was purified from an agarose gel. DNA of plasmidpVW119 was digested with BamH I and Bcl I, and the gel purified fragmentof 546 bp was ligated to the 3430 bp fragment. An E. coli transformantwas identified that harbored a plasmid that generated fragments of 3430and 546 upon digestion with BamH I and Bcl I. This plasmid was named pSGAdhA1.

The DNA of pSG AdhA1 was digested with Hind III and with Stu I. The endswere made flush by T4 DNA polymerase treatment and then ligated. An E.coli transformant that harbored a plasmid lacking Hind III and Stu Isites was identified and the DNA structure was verified by sequenceanalysis. The plasmid was named pSG AdhA1Δ. In this construct, 344 bp ofDNA had been deleted from the interior of the intron 1. The loss ofthese bases did not affect splicing of this intron. The functionalintron sequences ware obtained on a 213 bp fragment following digestionwith Bcl I and BamH I.

DNA of plasmid pCPL-Bgl was digested with Bgl II, and the linearized DNAwas ligated to the 213 bp Bcl I/BamH I fragment containing the deletedversion of the Adh1.S intron 1 sequences from pSG AdhA1Δ. An E. colitransformant was identified by restriction enzyme site mapping thatharbored a plasmid that contained the intron sequences ligated into theBgl II site, in the orientation such that the Bgl II/Bcl I juncture wasnearest the 5′ end of the MSV CPL leader sequence, and the Bgl II/BamH Ijuncture was nearest the 3′ end of the CPL. This orientation wasconfirmed by DNA sequence analysis. The plasmid was named pCPL A1I1Δ.The MSV leader/intron sequences was obtained from this plasmid bydigestion with BamH I and Nco I, and purification of the 373 bpfragment.

Construction of plant expression vectors based on the enhanced 35Spromoter, the MSV CPL, and the deleted version of the Adh1 intron 1 wasas follows. DNA of plasmid p35S En²/NOS was digested with BamH I, andthe 3562 bp linear fragment was ligated to a 171 bp fragment preparedfrom pMSV CPL DNA digested with BamH I. This fragment contained theentire MSV CPL sequence. An E. coli transformant was identified byrestriction enzyme site mapping that harbored a plasmid that containedthese sequences in an orientation such that the Nco I site waspositioned near the NOS Poly A sequences. This plasmid was named p35SEn² CPL/NOS. It contained the enhanced version of the 35S promoterdirectly contiguous to the MSV leader sequences such that the derivedtranscript included the MSV sequences in its 5′ untranslated portion.

The DNA of plasmid pKA882 was digested with Hind III and Nco I, and thelarge 4778 bp fragment was ligated to an 802 bp Hind III/Nco I fragmentcontaining the enhanced 35S promoter sequences and MSV leader sequencesfrom p35S En² CPL/NOS. An E. coli transformant harboring a plasmid thatcontained fragments of 4778 and 802 bp following digestion with Hind IIIand Nco I was identified, and named pDAB310. In this plasmid, theenhanced version of the 35S promoter was used to control expression ofthe GUS gene. The 5′ untranslated leader portion of the transcriptcontains the leader sequence of the MSV coat protein gene.

DNA of plasmid pDAB310 was digested with Nco I and Sac I. The large 3717bp fragment was purified from an agarose gel and ligated tocomplementary synthetic oligonucleotides. These oligonucleotides, whenannealed into double stranded structures, generated molecules havingsticky ends compatible with those left by Sac I, on one end of themolecule, and with Nco I on the other end of the molecule. In additionto restoring the sequences of the recognition sites for these twoenzymes, new sites were formed for the enzymes Kpn I, Xho I, and Hpa I.An E. coli transformant was identified that harbored a plasmid thatcontained sites for these enzymes, and the DNA structure was verified bysequence analysis. This plasmid was named pDAB1148.

DNA of plasmid pDAB1148 was digested with Bam HI and Nco I, the large3577 bp fragment was purified from an agarose gel and ligated to a 373bp fragment purified from pCPL A1I1Δ following digestion with Bam HI andNco I. An E. coli transformant was identified that harbored a plasmidthat generated fragments of 3577 and 373 bp following digestion withBamH I and Nco I, and the plasmid was named pDAB303. This plasmid hasthe following DNA structure: beginning with the base after the final Gresidue of the Pst I site of pUC19 (base 435), and reading on the strandcontiguous to the coding strand of the lacZ gene, linker sequence,nucleotides 7093 to 7344 of CaMV DNA, linker sequence, nucleotides 7093to 7439 of CaMV, linker sequence, nucleotides 167 to 186 of MSV,nucleotides 188 to 277 of MSV, a C residue followed by nucleotides 119to 209 of Adh1.S, nucleotides 555 to 672 of maize Adh1.S, linkersequence, nucleotides 278 to 317 of MSV, polylinker sequence containingrecognition sites for Hpa I, Xho I, Kpn I, and Sac I, nucleotides 1298to 1554 of NOS, and a G residue followed by the rest of the pUC19sequence (including the EcoR I site).

DNA of plasmid pDAB303 was digested with Nco I and Sac I, and the 3939bp fragment was ligated to the 1866 bp fragment containing the GUScoding region prepared from similarly digested DNA of pKA882. Theappropriate plasmid was identified by restriction enzyme site mapping,and was named pDAB305. This plasmid had the enhanced promoter, MSVleader and Adh1 intron arrangement of pDAB303, positioned to controlexpression of the GUS gene.

The plasmid pDAB305 contained an enhanced 35S promoter with additional3′ sequences and embodied as nucleotides 7093 to 7344 of CaMV DNA,linker sequence, nucleotides 7093 to 7439 of CaMV, linker sequence,nucleotides 167 to 186 of MSV, nucleotides 188 to 277 of MSV, a Cresidue followed by nucleotides 120 to 210 of maize Adh1.S, nucleotides555 to 672 of maize Adh1.S, linker sequence, nucleotides 278 to 317 ofMSV, and a G residue that represents the final base of an Nco Irecognition sequence. As above, the GUS translational start codon waspart of the Nco I site. Transcripts from this promoter contain as the 5′untranslated leader essentially the MSV coat protein leader sequence,into which has been inserted a deleted version of the maize Adh1.Sintron 1.

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 1 <210> SEQ ID NO 1 <211> LENGTH: 706<212> TYPE: DNA <213> ORGANISM: Agrobacterium tumefaciens<400> SEQUENCE: 1ctacaggcca aattcgctct tagccgtaca atattactca ccggtgcgat gc#cccccatc     60gtaggtgaag gtggaaatta atgatccatc ttgagaccac aggcccacaa ca#gctaccag    120tttcctcaag ggtccaccaa aaacgtaagc gcttacgtac atggtcgata ag#aaaaggca    180atttgtagat gttaacatcc aacgtcgctt tcagggatcc cgaattccaa gc#ttgggctg    240caggtcaatc ccattgcttt tgaagcagct caacattgat ctctttctcg ag#ggagattt    300ttcaaatcag tgcgcaagac gtgacgtaag tatccgagtc agtttttatt tt#tctactaa    360tttggtcgtt tatttcggcg tgtaggacat ggcaaccggg cctgaatttc gc#gggtattc    420tgtttctatt ccaacttttt cttgatccgc agccattaac gacttttgaa ta#gatacgct    480gacacgccaa gcctcgctag tcaaaagtgt accaaacaac gctttacagc aa#gaacggaa    540tgcgcgtgac gctcgcggtg acgccatttc gccttttcag aaatggataa at#agccttgc    600ttcctattat atcttcccaa attaccaata cattacacta gcatctgaat tt#cataacca    660 atctcgatac accaaatcga ctctagaact agtggatccg tcgacc   #                706  1

What is claimed is:
 1. A method for producing a fertile transgenicGossyphium hirsutum plant comprising the steps of: (i) establishing aregenerable callus culture from a Gossyphium hirsutum plant to betransformed wherein said callus culture is selected from the groupconsisting of embryogenic, hypocotyl-derived, and embryogenic,cotyledon-derived callus culture; (ii) selecting a plant cell aggregatetherefrom for transformation; (iii) transforming said plant cellaggregate with DNA by whisker-mediated transformation; (iv) identifyingtransformed cell lines therefrom; and (iv) regenerating fertiletransgenic plants therefrom.
 2. The method of claim 1 wherein saidculture is embryogenic, hypocotyl-derived.
 3. The method of claim 1wherein said culture is embryogenic, cotyledon-derived.
 4. The method ofclaim 1 wherein said culture is initiated on solid medium.